1. Field of the Invention
This invention provides methodology for in vitro selection and, if desired, subsequent identification of proteins or peptides with desired properties from pools of protein or peptide variants (libraries).
2. Discussion of the Related Art
Proteins and peptides, hereinafter jointly referred to as polypeptides, with desired properties such as binding affinity to a particular target molecule, catalytic activity, chemical or enzymatic activity or immunogenic activity are of great importance in many areas of biotechnology such as drug and vaccine development, diagnostic applications and bioseparation.
Recent progress in gene technology has provided the introduction of novel principles of isolating and identifying such polypeptides from large collections of variants constructed by different methods including combinatorial principles (Clackson and Wells, Trends Biotechnol. 12, pp. 173–184 [1994]). Typically, using biosynthesis for production of the library members, large pools of genes are constructed, encoding the individual library members, allowing for later selection or enrichment of desired variants using an appropriate bait molecule or chemical condition (Smith and Petrenko, Chem. Rev. 97, pp. 391–410 [1997]). For identification of selected variants, several techniques have been described to provide a physical link between the translated protein (phenotype) and the genetic information encoding it (genotype), allowing for identification of selected library members using DNA sequencing technology.
Using phage or cell display technologies, a genotype-phenotype coupling is obtained through incorporation of the individual library members into the coat or cell surface structures respectively of phage or cells containing the corresponding gene, which is typically inserted into phage, phagemid, plasmid or viral DNA. In the construction of such libraries, the gene pools need to be transformed into a recipient cell used for biosynthesis of the corresponding proteins. The practical limitations associated with this critical step to obtain large (complex) libraries (typically above 109 different members) have been a driving force for the development of alternative technologies based on in vitro transcription and translation of genetic information, thereby avoiding the transformation step.
Examples of such technologies are ribosomal display (Mattheakis et al., Proc. Natl. Acad. Sci. USA 91, pp. 9022–9026 [1994]; Hanes et al., FEBS Letters 450, pp. 105–110 [1999]) and RNA-peptide fusions using puromycin (Roberts and Szostak, Proc. Natl. Acad. Sci. USA 94, pp. 12297–12302 [1997]). In ribosomal display, a gene pool (typically polymerase chain reaction (PCR) products containing signals necessary for transcription and translation) is transcribed in vitro to produce a corresponding pool of mRNA used for ribosome mediated translation of proteins which typically, through the absence of translational stop signals, remain physically linked to the ribosome-mRNA complex. This allows for selection of polypeptides on the basis of the characteristics of the same and identification through DNA sequencing after conversion of the ribosome-associated mRNA into DNA by the use of reverse transcriptase. However, special precautions (temperature, buffer conditions) must be taken to ensure the stability of the ribosome-mRNA-protein complexes, limiting the conditions under which selection can be performed (Jermutus et al., Curr. Opin. Biotechnol. 9, pp. 534–548 [1998]; Hanes et al., op. cit. [1999]). In the RNA-peptide fusion system, puromycin-tagged RNA is used during translation, resulting in covalent RNA-protein/peptide links via acceptance by the ribosome of puromycin in the nascent polypeptide chain. However, new puromycin-mRNA fusions have to be prepared for each round of selection, severely limiting the efficiency of the technology (Jermutus et al., op. cit. [1998]; Roberts, Curr. Opin. Chem. Biol. 3, pp. 268–273 [1999]).
A further system has been described by Tawfik and Griffiths (Nature Biotechnology, (1998) 16; 652–656) which is cell free but seeks to mimic the effect of cells in creating compartments to link genotype and phenotype. Micelles are formed using a water-in-oil emulsion which can then be broking by mixing with ether. However, this system is not without problems, the two phase system results in several practical limitations. In order to recover the encapsulated molecules, the two phase system must be broken which is rather laborious, requiring several washes and causing a loss of material. Furthermore, the non-water components necessary to create the two-phase system might inhibit or denature biomolecules and the encapsulation itself makes it more difficult to deliver additional reagents necessary for e.g. detection or capture of specific molecular entities.